Modified lentiviral vector system for efficient delivery and long-term gene expression in human dendritic cells

Dendritic cells (DC) contain a high concentration of the antiviral factor SAMHD1, which inhibits the replication of lentiviruses. Some representatives of monkey lentiviruses, such as SIVmac, encode the accessory protein Vpx, which is an antagonist of SAMHD1 and blocks its action in infected cells to increase the efficiency of replication. However, the human lentivirus HIV-1, which is the basis for the vast majority of vector systems for the introduction and expression of genes, does not encode Vpx and infects DC with low efficiency. Based on this, it has been suggested that packaging of the SIVmac Vpx protein into HIV-1 lentiviral vector virions may increase their infectivity on DC. To enable the HIV-1-based lentiviral vector system to package the Vpx protein of SIVmac239 into virions, a number of modifications were introduced into the vector system. Based on the analysis of the structural and functional homology of the proteins p6 HIV-1 and SIVmac239, amino acid sequences were identified, the replacement of which in the composition of p6 HIV-1 would allow specific packaging of the exogenous Vpx protein SIVmac239, and have a minimal effect on the structure of the protein. To express the SIVmac239 Vpx protein in human cells, a codon-optimized sequence of the Vpx gene was obtained. The results showed that packaging of the Vpx protein into the virions of modified lentiviruses is critical for their infectivity on DC, since the “wild-type” virus, even obtained in the presence of the Vpx protein, was unable to productively infect DC because it does not contain the protein determinants for packaging Vpx. The efficiency of introducing a marker gene into DC using the modified vector system for different donors was 90–97 %. A distinctive feature of the vector system is its ability to package the SIVmac Vpx protein directly into HIV-1 virions, which makes it possible to directly introduce HIV-1-based viral vectors into DC without pre-treatment with the simian SIVmac virus. Thus, the modified vector system can be used for efficient transfer and long-term gene expression in human DC.

1. Steinman R. M. Decisions about dendritic cells: past, present, and future. Annual Review of Immunology, 2012, vol. 30, pp. 1–22. https://doi.org/10.1146/annurev-immunol-100311-102839

2. Steinman R. M, Banchereau J. Taking dendritic cells into medicine. Nature, 2007, vol. 449, pp. 419–426. https://doi. org/10.1038/nature06175

3. Tacken P. J., Zeelenberg I. S., Cruz L. J., van Hout-Kuijer M. A., van de Glind G., Fokkink R. G., Lambeck A. J. A., Figdor C. G. Targeted delivery of TLR ligands to human and mouse dendritic cells strongly enhances adjuvanticity. Blood, 2011, vol. 118, no. 26, pp. 6836–6844. https://doi.org/10.1182/blood-2011-07-367615

4. Naldini L. Lentiviruses as gene transfer agents for delivery to non-dividing cells. Current Opinion in Biotechnology, 1998, vol. 9, no. 5, pp. 457–463. https://doi.org/10.1016/s0958-1669(98)80029-3

5. Lahouassa H., Daddacha W., Hofmann H., Ayinde D., Logue E. C., Dragin L., Bloch N., Maudet C., Bertrand M., Gramberg T., Pancino G., Priet S., Canard B., Laguette N., Benkirane M., Transy C., Landau N. R., Kim B., Margottin-Goguet F. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nature Immunology, 2012, vol. 13, pp. 223–228. https://doi.org/10.1038/ni.2236

6. Laguette N., Sobhian B., Casartelli N., Ringeard M., Chable-Bessia C., Ségéral E., Yatim A., Emiliani S., Schwartz O., Benkirane M. SAMHD1 is the dendriticand myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature, 2011, vol. 474, pp. 654–657. https://doi.org/10.1038/nature10117

7. Hrecka K., Hao C., Gierszewska M., Swanson S. K., Kesik-Brodacka M., Srivastava S., Florens L., Washburn M. P., Skowronski J. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature, 2011, vol. 474, pp. 658–661. https://doi.org/10.1038/nature10195

8. Goujon C., Jarrosson-Wuillème L., Bernaud J., Rigal D., Darlix J. L., Cimarelli A. With a little help from a friend: increasing HIV transduction of monocyte-derived dendritic cells with virion-like particles of SIVMAC. Gene Therapy, 2006, vol. 13, pp. 991–994. https://doi.org/10.1038/sj.gt.3302753

9. Manel N., Hogstad B., Wang Ya., Levy D. E., Unutmaz D., Littman D. R. A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells. Nature, 2010, vol. 467, pp. 214–217. https://doi.org/10.1038/nature09337

10. Sambrook J. Molecular cloning: a laboratory manual, 3d ed. Cold Spring Harbor, 2001.

11. Accola M. A., Bukovsky A. A., Jones M. S., Göttlinger H. G. A conserved dileucine-containing motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency viruses SIV(mac) and SIV(agm). Journal of Virology, 1999, vol. 73, no. 12, pp. 9992–9999. https://doi.org/10.1128/JVI.73.12.9992-9999.1999

12. Fossen T., Wray V., Bruns K., Rachmat J., Henklein P., Tessmer U., Maczurek A., Klinger P., Schubert U. Solution structure of the human immunodeficiency virus type 1 p6 protein. Journal of Biological Chemistry, 2005, vol. 280, no. 52, pp. 42515–42527. https://doi.org/10.1074/jbc.M507375200

13. Zhai Q., Landesman M. B., Robinson H., Sundquist W. I., Hill C. P. Identification and structural characterization of the ALIX-binding late domains of simian immunodeficiency virus SIVmac239 and SIVagmTan-1. Journal of Virology, 2011, vol. 85, no. 1, pp. 632–637. https://doi.org/10.1128/JVI.01683-10

14. Kondo E., Göttlinger H. G. A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr. Journal of Virology, 1996, vol. 70, no. 1, pp. 159–164. https://doi. org/10.1128/JVI.70.1.159-164.1996.

15. Zhu H., Jian H., Zhao L. J. Identification of the 15FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion. Retrovirology, 2004, vol. 1, art. 26. https://doi.org/10.1186/1742-4690-1-26